Cryo-electron microscopy (cryo-EM) is a powerful technique involving the imaging of biological materials in a transmission electron microscope under cryogenic conditions. Cryo-EM may be used to study structures of radiation-sensitive specimen, such as biological macromolecules and complexes, which are embedded in a thin film of vitreous ice. Atomic resolution structures have been determined by cryo-EM, and all-atom models have been built. Cryo-EM has also been employed in other scientific fields such as material science (e.g., phase separation of blend polymers, self-assembled nanostructures), nanomedicine (e.g., nanostructured materials for intracellular delivery of agents), and renewable energy (e.g., nanostructured materials for solar cells).
In cryo-EM, one limiting factor in the image contrast, and thus the resolution of the determined structure, is the ice thickness. The thicker the ice layer is, the lower contrast the cryo-EM images have. Experiments have shown that by extensive efforts to optimize the vitrification process, the contrast of recorded cryo-EM images increased dramatically. Currently, the freezing process is still a trial and error method, and there is very poor control of the ice thickness. Typically, several cryo-samples are prepared on Transmission Electron Microscopy (TEM) grids under a variety of conditions, with the hope that one of those conditions will produce a vitrified sample having the desired ice thickness. Each of these samples has to be screened using cryo-EM in order to identify whether the sample has achieved the desired ice thickness. This process is time consuming, labor intensive, and is wasteful in that it results in the excessive usage of the TEM, and loss of the biological material in the unsuccessful samples. Moreover, despite the use of multiple samples, there is still no assurance that the desired thickness will be achieved, due to the unpredictability of the process.
Accordingly, there is a need for improved systems and methods for cryo-EM.